Immunogenic Constructs

ABSTRACT

The present invention relates to molecules, which can be used to induce a therapeutic or prophylactic immune response against MAP. In particular, the present invention relates to polypeptides comprising an alipC polypeptide sequence, a gsd polypeptide sequence, a p12 polypeptide sequence and an mpa polypeptide sequence, wherein said ahpC polypeptide comprises the sequence of SEQ ID NO: 2, a variant thereof having more than 70% amino acid sequence identity to SEQ ID NO: 2 across the full length of SEQ ID NO: 2, or a fragment of at least 8 amino acids of SEQ ID NO: 2 which comprises an epitope; said gsd polypeptide comprises the sequence of SEQ ID NO: 6, a variant thereof having more than 70% amino acid sequence identity to SEQ ID NO: 6 across the full length of SEQ ID NO: 6, or a fragment of at least 8 amino acids of SEQ ID NO: 6 which comprises an epitope; said pi 2 polypeptide comprises the sequence of SEQ ID NO: 10, a variant thereof having more than 70% amino acid sequence identity to SEQ ID NO: 10 across the full length of SEQ ID NO: 10, or a fragment of at least 8 amino acids of SEQ ID NO: 10 which comprises an epitope; and said mpa polypeptide comprises the sequence of SEQ ID NO: 14, a variant thereof having more than 70% amino acid sequence identity to SEQ ID NO: 14 across the full length of SEQ ID NO: 14, or a fragment of at least 8 amino acids of SEQ ID NO: 14 which comprises an epitope. Preferably such a variant maintains the ability to generate an immune response against the unmodified polypeptide.

FIELD OF THE INVENTION

The present invention relates to the treatment or prevention ofinfection with Mycobacterium avium subspecies paratuberculosis (MAP),and to the treatment or prevention of disorders associated with suchinfection.

BACKGROUND TO THE INVENTION

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of theMycobacterium avium complex MAC. Unlike other environmental MAC, MAP hasthe specific ability to cause chronic inflammation of the intestine of arange of histopathological types in many animals including primates.Despite its broad pathogenicity, MAP can live in animals for yearswithout causing clinical disease. MAP is more thermotolerant than M.bovis and has been cultured from retail pasteurised milk in the UK,Czech Republic and the USA. Transmittal from livestock to humans by thisroute is therefore probable. There is also a high risk of transmittalfrom sources of environmental contamination such as rivers and surfacewaters used for domestic supply.

MAP can cause of Crohn's disease in humans, in particular in people whohave an inherited or acquired susceptibility. Recent studies haveconfirmed that the inflamed intestine of most people with Crohn'sdisease is infected with these chronic enteric pathogens. Furtherstudies have reported that MAP can be cultured from the blood of 50% ofpatients with Crohn's disease showing that, as in animals, the infectionis often systemic. Furthermore a high proportion of people withIrritable Bowel Syndrome are also infected with MAP.

The organisms in humans are very slow growing and exceedingly difficultto isolate and passage in conventional culture. They are present in lowabundance and adopt a Ziehl Neelsen (ZN) staining negative form whichcannot be seen in tissues by ordinary light microscopy. They appear tobe able to minimise immune recognition and unlike conventionalspheroplasts, their ZN negative form is highly resistant to chemical andenzymatic lysis procedures essential for reliable detection by PCR.

MAP infections are extremely difficult to eradicate. ZN-negativeintracellular MAP are highly resistant in vivo to standard anti-TBdrugs. However, a substantial proportion of patients with Crohn'sdisease who can take rifabutin/clarithromycin combinations, to which MAPare more sensitive, heal, sometimes dramatically.

SUMMARY OF THE INVENTION

The present invention relates to molecules, in particular polypeptidesand the polynucleotides which can be used to express them, which can beused to induce a therapeutic or prophylactic immune response against MAPin humans and animals.

In particular, the present invention provides a polypeptide comprisingan ahpC polypeptide sequence, a gsd polypeptide sequence, a p12polypeptide sequence and an mpa polypeptide sequence, wherein

said ahpC polypeptide comprises the sequence of SEQ ID NO: 2, a variantthereof having more than 70% amino acid sequence identity to SEQ ID NO:2 across the full length of SEQ ID NO: 2, or a fragment of at least 8amino acids of SEQ ID NO: 2 which comprises an epitope;

said gsd polypeptide comprises the sequence of SEQ ID NO: 6, a variantthereof having more than 70% amino acid sequence identity to SEQ ID NO:6 across the full length of SEQ ID NO: 6, or a fragment of at least 8amino acids of SEQ ID NO: 6 which comprises an epitope;

said p12 polypeptide comprises the sequence of SEQ ID NO: 10, a variantthereof having more than 70% amino acid sequence identity to SEQ ID NO:10 across the full length of SEQ ID NO: 10, or a fragment of at least 8amino acids of SEQ ID NO: 10 which comprises an epitope; and

said mpa polypeptide comprises the sequence of SEQ ID NO: 14, a variantthereof having more than 70% amino acid sequence identity to SEQ ID NO:14 across the full length of SEQ ID NO: 14, or a fragment of at least 8amino acids of SEQ ID NO: 14 which comprises an epitope.

Preferably such a variant maintains the ability to generate an immuneresponse against the unmodified polypeptide. A preferred variant ahpCpolypeptide has the amino acid sequence given in SEQ ID NO: 4. Apreferred variant gsd polypeptide has the amino acid sequence given inSEQ ID NO: 8. A preferred variant p12 polypeptide has the amino acidsequence given in SEQ ID NO: 12. A preferred variant mpa polypeptide hasthe amino acid sequence given in SEQ ID NO: 16. A preferred polypeptideof the invention comprises the amino acid sequence of SEQ ID Nos: 4, 8,12 and 16. A particularly preferred amino acid sequence is given in SEQID NO: 24.

The present invention also provides polynucleotides which encode suchpolypeptides. A polynucleotide of the invention may comprise

(a) the ahpC polynucleotide of SEQ ID NO: 1 or a variant thereof havingat least 70% homology to SEQ ID NO: 1 across the full length of SEQ IDNO: 1 or a fragment of at least 24 nucleotides of SEQ ID NO: 1 whichencodes an epitope;

(b) the gsd polynucleotide of SEQ ID NO: 5 or a variant thereof havingat least 70% homology to SEQ ID NO: 5 across the full length of SEQ IDNO: 5 or a fragment of at least 24 nucleotides of SEQ ID NO: 5 whichencodes an epitope;

(c) the p12 polynucleotide of SEQ ID NO: 9 or a variant thereof havingat least 70% homology to SEQ ID NO: 1 across the full length of SEQ IDNO: 9 or a fragment of at least 24 nucleotides of SEQ ID NO: 9 whichencodes an epitope; and

(d) the mpa polynucleotide of SEQ ID NO: 13 or a variant thereof havingat least 70% homology to SEQ ID NO: 1 across the full length of SEQ IDNO: 13 or a fragment of at least 24 nucleotides of SEQ ID NO: 13 whichencodes an epitope.

A polynucleotide of the invention will encode a polypeptide of theinvention. In one embodiment a polynucleotide variant as defined abovewill differ from the given SEQ ID NO by virtue of degeneracy in thegenetic code. In one embodiment, a polynucleotide variant will be codonoptimised for the species which it is desired to treat.

The present invention also provides:

A vector comprising a polynucleotide of the invention, or a vectorcapable of expressing an ahpC polypeptide, a gsd polypeptide, a p12polypeptide and an mpa polypeptide as defined above. Preferred vectortypes include poxvirus vectors, adenovirus vectors and plasmids.

A host cell comprising a polypeptide, polynucleotide or vector of theinvention or a host cell capable of expressing a polypeptide of theinvention.

A polypeptide, polynucleotide, vector or host cell of the invention foruse in therapy. In particular, the use of such a polypeptide,polynucleotide, vector or host cell in the manufacture of a medicamentfor treating or preventing MAP infection or a condition or symptomassociated with MAP infection.

A method of treating or preventing MAP infection of a condition orsymptom associated with MAP infection comprising administering to asubject in need thereof an effective amount of a polypeptide,polynucleotide, vector or host cell of the invention. The subject mayalso be administered with a further therapeutic agent.

A kit for use in treating or preventing MAP infection or a condition orsymptom associated with MAP infection, said kit comprising (i) at leastone polypeptide, polynucleotide, vector or host cell of the inventionand (ii) at least one other therapeutic agent, for simultaneous,sequential or separate use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide and amino acid sequences of the Havilahconstruct (SEQ ID NOs:26 and 27). The amino acid sequence comprises aubiquitin leader sequence followed by an ahpC sequence (italics), a gsdsequence (bold), a p12 sequence (plain text) and an mpa sequence (bolditalics). The amino acid sequence ends with a pK tag.

FIG. 2 shows the ahpC (A) (SEQ ID NO:4), gsd (B) (SEQ ID NO:8), p12 (C)(SEQ ID NO:2) and mpa (D) (SEQ ID NO:16) polypeptides included in theHavilah construct. Bold italics=predicted strong class II human epitope.Underlined=predicted class I epitope.

FIG. 3A shows the highly significantly increased antigen-specificELISPOT responses to rec.AhpC and rec.Mpa, as well as to syntheticpeptides from the Havilah polyprotein in pools B and F, resulting fromvaccination of naïve uninfected C57/BL6 MICE with the recombinantplasmid pSG2.HAV followed by MVA.HAV when compared to vector-onlycontrols. B. example of the identification of an epitope from ELISPOTresponses to the synthetic peptides in pool F (see also FIG. 4) in thepSG2.HAV/MVA.HAV vaccinated animals. The response in vaccinated, but notunvaccinated animals is seen to be due to the strong recognition of thepeptide 9.1 with the amino acid sequence GFAEINPIA constituting aspecific T cell epitope.

FIG. 4. Summary of the sequences of the synthetic peptide antigensspanning the Havilah polyprotein used in the detection of epitoperegions (SEQ ID NOs:28-167). AhpC peptides in italics, Mpa peptides inbold italics, p12 peptides in plain text, and gsd peptides in bold.

FIG. 5A is a diagram of the structure of the mpa antigen in the cellsurface membrane of MAP showing intracellular, transmembrane andextracellular domains. The strong T cell epitope GFAEINPIA (SEQ IDNO:25) in peptide 9.1 is seen to comprise the fifth and smallestextracellular loop of the protein. B. Highly significant reduction by2-3 log units and elimination of MAP organisms in the spleen tissue ofMAP-infected C57/BL6 mice in response to therapeutic vaccination, 26weeks after infection with a slow growing laboratory strain of MAPcompared to vector-only and sham vaccinated animals. *MAP not detectedby sensitive qPCR in the spleens of 6 of the 8 mice.

FIG. 6. Highly significant antigen-specific T cell (A. ELISPOT) andantibody (B. ELISA) responses to vaccination using Ad5.HAV to primefollowed by MVA.HAV to boost in C57/BL6 mice. Prominent T cellrecognition of the strong epitope in peptide 9.1 is again seen togetherwith significant recognition of rec.Gsd and rec.Mpa proteins and peptidepools J and L, compared with animals vaccinated using vectors alone. Bycontrast none of the peptides including 9.1 are recognised by antibodyin ELISA assays in response to vaccination but there is substantialrecognition of rec.AhpC and rec.Mpa.

FIG. 7. Highly significant reduction compared to vector-only controls,in the infective load of MAP organisms in the spleen tissue of C57/BL6mice challenged i.p with low dose or high dose MAP in response tovaccination using Ad5.HAV followed by MVA.HAV given either before(prophylactic) or after (therapeutic) the MAP challenge. A=prophylactictreatment (E1+G1). Low dose vector vs vaccine: p=0.0207; high dosevector vs vaccine: p=0.0205. B=therapeutic treatment (E2+G2). Low dosevector vs vaccine: p=0.0207; high dose vector vs vaccine: p=0.0023.

FIG. 8. The same experiment as in FIG. 7 also showing significantreductions in the MAP infective load in the liver tissues of micevaccinated with Ad5.HAV followed by MVA.HAV as described above.A=prophylactic treatment (E1+G1). Low dose vector vs vaccine: p=0.0379;high dose vector vs vaccine: p=0.0003. B=therapeutic treatment (E2+G2).Low dose vector vs vaccine: p=0.0499.

FIG. 9. Prominent recognition of the strong T cell epitope GFAEINPIA inmpa (peptide 9.1) and significant recognition of peptide pool J comparedto vector-only controls, in the presence of MAP infection in C57/BL6mice, in response to vaccination administered prophylactically (A) ortherapeutically (B) as described for FIG. 7 above. In the presence ofestablished MAP infection (therapeutic panel B) there is alsosignificant recognition of peptide pool L compared to vector-onlycontrols such that pre-existing MAP infection was capable of priming theresponse to vaccination. A=Pool J (p=0.0006); peptide 9.1 (p=<0.0001).B=Pool J (p=<0.0001); Pool L (p=0.0032); Peptide 9.1 (p=<0.0001).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID Nos 1 and 2 are the polynucleotide and polypeptide sequencesrespectively for the MAP ahpC gene.

SEQ ID Nos 3 and 4 are a modified version of the MAP ahpC gene, in whichthe polynucleotide sequence has been codon optimised for human use.

SEQ ID Nos 5 and 6 are the polynucleotide and polypeptide sequencesrespectively for the MAP gsd gene.

SEQ ID Nos 7 and 8 are a modified version of the MAP gsd gene, in whichthe polynucleotide sequence has been codon optimised for human use andwhich is truncated at the N-terminal in order to remove the cysteineresidue at position 22.

SEQ ID Nos 9 and 10 are the polynucleotide and polypeptide sequencesrespectively for the MAP p12 gene.

SEQ ID Nos 11 and 12 are a modified version of the MAP p12 gene, inwhich the polynucleotide sequence has been codon optimised for humanuse.

SEQ ID Nos 13 and 14 are the polynucleotide and polypeptide sequencesrespectively for the MAP mpa gene.

SEQ ID Nos 15 and 16 are a modified version of the MAP mpa gene, inwhich the polynucleotide sequence has been codon optimised for human useand a number of transmembrane regions have been removed.

SEQ ID NO: 17 is a ubiquitin leader sequence.

SEQ ID NO: 18 is a pK Tag sequence.

SEQ ID Nos 19 and 20 are a polynucleotide construct and the encodedpeptide consisting of the modified ahpC, gsd, p12 and mpa sequencesabove.

SEQ ID Nos 21 and 22 are a polynucleotide construct and the encodedpeptide consisting of the modified ahpC, gsd, p12 and mpa sequencesabove, together with a ubiquitin leader sequence and a pK tag.

SEQ ID NO: 23 is the polynucleotide sequence of the Havilah constructand SEQ ID NO: 24 is the polypeptide sequence encoded by Havilah.

SEQ ID NO: 25 is a T cell epitope from the mpa polypeptide sequence asidentified in FIGS. 3 and 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a combination of fourproteins deriving from MAP in the treatment or prevention of MAPinfection or conditions associated with the presence of MAP in humans oranimals. The four proteins are ahpC, gsd, p12 and mpa. AhpC and p12 aresecreted components while gsd and mpa are both membrane bound moleculesin MAP.

ahpC is a secreted component shared by many pathogenic mycobacteria. Itis involved in the ability of MAP to survive within macrophages and isupregulated on entry into a state of microbial dormancy. The nucleicacid and amino acid sequences of the MAP ahpC gene and protein are givenin SEQ ID Nos 1 and 2 respectively. For use in the present invention,this sequence, or a variant thereof as discussed below may be used. Forexample, the MAP ahpC gene sequence may be codon optimised as discussedfurther below to make it more suitable for mammalian, in particularhuman, use. A suitable modified ahpC sequence and encoded protein aregiven in SEQ ID Nos 3 and 4 respectively.

gsd is a glycosyl transferase encoded by the GS pathogenicity elementwith a predicted signal sequence and lipid acylation site. Microarrayanalysis shows that it is up-regulated in the intracellular environment.It is expressed on the microbial cell surface and is predicted totransfer GDP-fucose to sub-terminal rhamnose to cap surfaceglycopeptidolipid on MAP with derivatised fucose giving the pathogen inits ZN-negative state an inert, hydrophobic, and highly resistant cellsurface. The nucleic acid and amino acid sequences of the MAP gsd geneand protein are given in SEQ ID Nos 5 and 6 respectively. For use in thepresent invention, this sequence, or a variant thereof as discussedbelow, may be used. For example, the MAP gsd gene sequence may be codonoptimised as discussed further below to make it more suitable formammalian, in particular human, use. Other modifications may be made,for example potential acylation sites may be removed. One suitablemodified gsd sequence and encoded protein are given in SEQ ID Nos 7 and8 respectively.

p12 is the carboxyterminal 17 kDa fragment of p43 encoded by IS900 whichis also up-regulated intracellularly. It is strongly predicted on thecell surface and both in MAP and in p43.rec.E. coli it is the substratefor specific proteolytic cleavage and exodomain release. The nucleicacid and amino acid sequences of the MAP p12 gene and protein are givenin SEQ ID Nos 9 and 10 respectively. For use in the present invention,this sequence, or a variant thereof as discussed below may be used. Forexample, the MAP p12 gene sequence may be codon optimised as discussedfurther below to make it more suitable for mammalian, in particularhuman, use. One suitable modified p12 sequence and encoded protein aregiven in SEQ ID Nos 11 and 12 respectively.

mpa is also expressed on the surface of MAP and is believed to be uniqueto the pathogen. It is both an acetylase and a predicted pore moleculewith 10 transmembrane regions and a large extracellular peptide loop.The nucleic acid and amino acid sequences of the MAP mpa gene andprotein are given in SEQ ID Nos 13 and 14 respectively. For use in thepresent invention, this sequence, or a variant thereof as discussedbelow may be used. For example, the MAP mpa gene sequence may be codonoptimised as discussed further below to make it more suitable formammalian, in particular human, use. Other modifications may be made,for example transmembrane regions may be removed to reduce thehydrophobicity of the protein. One suitable modified mpa sequence andencoded protein are given in SEQ ID Nos 15 and 16 respectively.

According to the present invention each of these four proteins, orvariants of any thereof, are provided in combination.

Polypeptides

The invention relates to the provision of an ahpC polypeptide, a gsdpolypeptide, a p12 polypeptide and an mpa polypeptide in combination.

A “polypeptide” is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The term “polypeptide” thus includes shortpeptide sequences and also longer polypeptides and proteins. As usedherein, the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including glycine and both the D or L opticalisomers, and amino acid analogs and peptidomimetics.

A suitable ahpC polypeptide may have the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. A suitable gsd polypeptide may have the aminoacid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. A suitable p12polypeptide may have the amino acid sequence of SEQ ID NO: 10 or SEQ IDNO: 12. A suitable mpa polypeptide may have the amino acid sequence ofSEQ ID NO: 14 or SEQ ID NO: 16. A suitable ahpC, gsd, p12 or mpasequence may alternatively be a variant of one of these specificsequences. For example, a variant may be a substitution, deletion oraddition variant of any of the above amino acid sequences, or may be afragment of any thereof as described further below.

A variant of one of the four polypeptide may comprise 1, 2, 3, 4, 5, upto 10, up to 20, up to 30 or more amino acid substitutions and/ordeletions from the sequences given in the sequence listing. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Substitution” variants preferablyinvolve the replacement of one or more amino acids with the same numberof amino acids and making conservative amino acid substitutions. Forexample, an amino acid may be substituted with an alternative amino acidhaving similar properties, for example, another basic amino acid,another acidic amino acid, another neutral amino acid, another chargedamino acid, another hydrophilic amino acid, another hydrophobic aminoacid, another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as follows:

Ala aliphatic, hydrophobic, Met hydrophobic, neutral neutral Cys polar,hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar,hydrophilic, charged Pro hydrophobic, neutral (−) Glu polar,hydrophilic, charged Gln polar, hydrophilic, neutral (−) Phe aromatic,hydrophobic, Arg polar, hydrophilic, charged neutral (+) Gly aliphatic,neutral Ser polar, hydrophilic, neutral His aromatic, polar, Thr polar,hydrophilic, neutral hydrophilic, charged (+) Ile aliphatic,hydrophobic, Val aliphatic, hydrophobic, neutral neutral Lys polar,hydrophilic, charged Trp aromatic, hydrophobic, (+) neutral Leualiphatic, hydrophobic, Tyr aromatic, polar, hydrophobic neutral

Preferred “derivatives” or “variants” include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatized or modified, e.g. labelled, providingthe function of the peptide is not significantly adversely affected.

Derivatives and variants as described above may be prepared duringsynthesis of the peptide or by post-production modification, or when thepeptide is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

Suitable variants may also be naturally occurring polypeptides frommycobacteria other than MAP. For example, a variant ahpC polypeptidesequence may derive from a different mycobacterial strain to MAP. Suchnaturally occurring variants preferably maintain the ability tostimulate an immune response which is capable of acting against MAP.That is, the immune response to the variant polypeptide will reactagainst MAP polypeptides as well as the variant polypeptide used.

Preferably variants according to the invention have an amino acidsequence which has more than 60%, or more than 70%, e.g. 75 or 80%,preferably more than 85%, e.g. more than 90 or 95% amino acid identityto, for example, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, (according tothe test described hereinafter). This level of amino acid identity maybe seen across the full length of the sequence or over a part of thesequence, such as 20, 30, 50, 75, 100, 150, 200 or more amino acids,depending on the size of the full length polypeptide.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:O, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatized.

Particular modifications can be made to any of the wild type MAPproteins sequences given in SEQ ID Nos: 2, 6, 10 and 14. For example,modification can be made to try to improve the overall properties of thevariant protein as an immunogen.

In one embodiment, a wild-type protein may be modified by deletion orsubstitution to remove an acylation site. Such an acylation site mightaffect the overall conformation of the protein. By omitting acylationsites, for example by excluding or substituting a cysteine residue, thepresentation of effective epitopes within the protein may be optimised.For example, the wild type MAP gsd sequence given in SEQ ID NO: 6includes a cysteine residue at position 22. In the variant of SEQ ID NO:8, the amino acid sequence has been modified by truncation at theN-terminal such that this cysteine residue is no longer present. Such amodification may be made to a wild type protein or to any of the variantor fragment sequences, such as the codon optimised sequences, describedherein.

In another embodiment, a wild type MAP protein may be modified todisable or remove potential cross-reacting epitopes. For example, wherea polypeptide of the invention is intended for use in a human, thepolypeptide sequence may be modified to disable or remove potentialcross-reacting human epitopes, such as sequences which generateantibodies in human patients which may cross-react with similarsequences in human proteins. Modifications may thus be made to the MAPsequences to avoid such cross-reactivity but to maintain the ability togenerate an anti-MAP immune response.

For example within the wild type MAP gsd sequence the lysine residues atpositions 239 and 241 (see SEQ ID NO: 6) may each be substituted withasparagine. An equivalent substitution may be made in any of the variantor fragment gsd sequences described herein. For example in the variantsequence of SEQ ID NO: 8, the lysine residues at positions 216 and 218may be replaced with asparagines. This may be achieved by modifying thenucleic acid sequence which encodes the gsd polypeptide. For example, inthe gsd polynucleotide sequence of SEQ ID NO: 7, the AAG codons atpositions 646 to 648 and 651 to 654 may be replaced by AAT. Thismaintains the optimised human codon usage of SEQ ID NO: 7 and furtherremoves potentially cross-reacting human epitopes.

Similarly, modifications mat be made to the MAP ahpC sequence. In thewild-type ahpC sequence of SEQ ID NO: 2, the lysine at position 29 maybe replaced with threonine and the proline at position 31 may bereplaced with leucine. An equivalent substitution may be made in any ofthe variant or fragment ahpC sequences described herein. For example, inthe modified variant sequence of SEQ ID NO: 4, the same substitutionsmay be made at the position 28 lysine and the position 30 proline. Thismay be achieved by modifying the nucleic acid sequence which encodes theahpC polypeptide. For example, in the ahpC polynucleotide sequence ofSEQ ID NO: 3, the AAA codon at positions 82 to 84 may be replaced by ACAand the CCC codon at positions 88 to 90 may be replaced by CTC. Thismaintains the optimised human codon usage of SEQ ID NO: 3 and furtherremoves potentially cross-reacting human epitopes.

Similarly, modifications may be made to reduce the hydrophobicity of theprotein and thus to help optimise the surface presentation of epitopes.For example, the wild-type mpa sequence of SEQ ID NO: 14 includes tentransmembrane regions. In order to reduce the hydrophobicity of theprotein, one or more of these regions, or parts of these regions, may beomitted or substituted. For example, one, more or all of thetransmembrane regions may be deleted. Such regions may be deletedtotally or partially, optionally leaving none, one, two or more aminoacid residues from the ends of the transmembrane sequence in theprotein. Thus one modification may be the deletion or substitution ofone or more hydrophobic amino acids. An example of this is seen in SEQID NO: 16 which is a variant of MAP mpa in which most of thetransmembrane sequences have been deleted, leaving only one or two aminoacids from the transmembrane regions in the variant polypeptide.

Polypeptide “fragments” according to the invention may be made bytruncation, e.g. by removal of one or more amino acids from the N and/orC-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40or more amino acids may be removed from the N and/or C terminal in thisway. Fragments may also be generated by one or more internal deletions.For example, a variant of the invention may consist of or comprise twoor more epitope regions from a full length polypeptide of the region inthe absence of non-epitope amino acids. Preferably a fragment of anahpC, gsd, p12 or mpa polypeptide comprises at least one epitope capableof inducing an immune response against the unmodified MAP polypeptide.Such fragments may be derived from a sequence of SEQ ID NO: 2, 4, 6, 8,10, 12, 14 or 16 or may be derived from a variant peptide as describedherein. Preferably such fragments are between 8 and 150 residues inlength, e.g. 8 to 50 or 8 to 30 residues. Alternatively, fragments ofthe invention may be longer sequences, for example comprising at least50%, at least 60%, at least 70%, at least 80% or at least 90% of a fulllength polypeptide of the invention.

Preferably, a variant of one of the four polypeptides is a functionalvariant thereof. In particular, a variant polypeptide should retain theability to stimulate an immune response against the unmodified MAPpolypeptide. In one embodiment, a functional variant polypeptide shouldbe capable of acting as an antigen and should include at least onefunctional epitope from the original polypeptide.

An “antigen” refers to any agent, generally a macromolecule, which canelicit an immunological response in an individual. As used herein,“antigen” is generally used to refer to a polypeptide molecule orportion thereof which contains one or more epitopes. Furthermore, forthe purposes of the present invention, an “antigen” includes apolypeptide having modifications, such as deletions, additions andsubstitutions (generally conservative in nature) to the native sequence,so long as the polypeptide maintains sufficient immunogenicity. Thesemodifications may be deliberate, for example through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the antigens.

An “immune response” against an antigen of interest is the developmentin an individual of a humoral and/or a cellular immune response to thatantigen. For purposes of the present invention, a “humoral immuneresponse” refers to an immune response mediated by antibody molecules,while a “cellular immune response” is one mediated by T-lymphocytesand/or other white blood cells.

As used herein, the term “epitope” generally refers to the site on atarget antigen which is recognised by an immune receptor such as aT-cell receptor and/or an antibody. Preferably it is a short peptidederived from or as part of a protein. However the term is also intendedto include peptides with glycopeptides and carbohydrate epitopes. Asingle antigenic molecule, such as one of the four proteins describedherein, may comprise several different epitopes. The term “epitope” alsoincludes modified sequences of amino acids or carbohydrates whichstimulate responses which recognise the whole organism.

It is advantageous if the selected epitope is specific to MAP, orinvolved in the pathogenicity of MAP. For example, it is advantageous ifthe immune receptor and/or antibody which recognises the epitope willonly recognise this epitope from MAP, and not epitopes in otherunrelated proteins, in particular proteins from unrelated organisms orhost proteins. If the epitope is involved in pathogenicity of MAP, thenan immune response against such an epitope may be used to targetpathogenic MAP infections.

An epitope may also be related to equivalent epitopes on othermycobacteria. For example, many individuals suffering from MAP infectionare also infected by M. avium as a secondary co-pathogen. Other M. aviumcomplexes may be present or involved in Crohn's disease. Many of theproteins expressed in MAP such as AhpC are very similar to thoseexpressed in M. avium. If the polypeptide of the invention includes oneor more epitopes which are capable of stimulating an immune responsewhich acts against M. avium in addition to MAP, a further, secondary,therapeutic effect may be achieved.

Epitopes can be identified from knowledge of the amino acid andcorresponding DNA sequences of the peptide or polypeptide, as well asfrom the nature of particular amino acids (e.g., size, charge, etc.) andthe codon dictionary, without undue experimentation. See, e.g., IvanRoitt, Essential Immunology, 1988; Janis Kuby, Immunology, 1992 e.g.,pp. 79-81. Some guidelines in determining whether a protein or anepitope of interest will stimulate a response, include: peptidelength—the peptide should be at least 8 or 9 amino acids long to fitinto the MHC class I complex and at least 8-25, such at least as 13-25amino acids long to fit into a class II MHC complex. These lengths arethe minimum for the peptide to bind to the respective MHC complex. It ispreferred for the peptides to be longer than these lengths because cellsmay cut peptides. The peptide should contain an appropriate anchor motifwhich will enable it to bind to the various class I or class IImolecules with high enough specificity to generate an immune response.This can be done, without undue experimentation, by comparing thesequence of the protein of interest with published structures ofpeptides associated with the MHC molecules. Thus, the skilled artisancan ascertain an epitope of interest by comparing the protein sequencewith sequences listed in the protein database.

Suitable epitopes may thus be identified by routinely used methods suchas those demonstrated in FIGS. 3 and 4 for identifying the strong T cellepitope GFAEINPIA (peptide 9.1) in the 5^(th) extracellular loop of mpa.In such a method, a library of short peptides which are fragments of thepolypeptide sequence of interested may be generated and each of thesepeptides assessed separately for their ability to identify an immuneresponse against the full length polypeptide. Members of the library maybe screened in groups or pools or individual members of the library,such as individual members of a single pool, may be assessed separately.

In a further example, epitope scanning of the individual proteins of SEQID Nos 4, 8, 12 and 16 revealed a number of predicted class I and classII epitopes.

In the ahpC variant sequence of SEQ ID NO: 4, predicted strong class IIepitopes were identified at amino acids 48 to 56, 90 to 101 and 161 to169. An ahpC polypeptide of the invention, such as an ahpC variant orfragment polypeptide, preferably comprises at least one, for exampleone, two or all three of these epitopes.

In the gsd variant sequence of SEQ ID NO: 8, predicted class I epitopeswere identified at amino acids 1 to 32, 58 to 68, 99 to 119, 123 to 147,159 to 169, 180 to 194 and 200 to 231, and predicted strong class IIepitopes were identified at amino acids 64 to 76, 95 to 110, 192 to 206and 223 to 240. A gsd polypeptide of the invention, such as a gsdvariant or fragment polypeptide, preferably comprises at least one, forexample one, two, three, four, five, six, seven, eight, nine, ten or allof these epitopes.

In the p12 variant sequence of SEQ ID NO 12, predicted class I epitopeswere identified at amino acids 33 to 56 and 98 to 117 and a predictedstrong class II epitope was identified at amino acids 3 to 10. A p12polypeptide of the invention, such as a p12 variant or fragmentpolypeptide, preferably comprises at least one, for example one, two orall three of these epitopes.

In the mpa variant sequence of SEQ ID NO: 16, a predicted class Iepitope was identified at amino acids 130 to 160, and predicted strongclass II epitopes were identified at amino acids 56 to 64 and 150 to160. An mpa polypeptide of the invention, such as an mpa variant orfragment polypeptide, preferably comprises at least one, for exampleone, two or all three of these epitopes.

As shown in the Examples, a particular strong T cell epitope has beenidentified in the mpa polypeptide sequence. This epitope has the aminoacid sequence GFAEINPIA (SEQ ID NO: 25) and is located at amino acids357 to 365 of SEQ ID NO: 14 and amino acids 177 to 185 of SEQ ID NO: 16.This sequence is found in the construct of SEQ ID NO: 24 at amino acids761 to 769. A preferred mpa polypeptide sequence is a sequence whichcomprises GFAEINPIA. Such a sequence may also comprise one, two or allthree of the predicted class I and class II epitopes mentioned above.

This epitope is believed to be located in the fifth extracellular loopof mpa (FIG. 5A). A preferred mpa polypeptide may therefore maintain thesequence of the fifth extracellular loop. An mpa polypeptide maytherefore comprise the amino acid sequence GFAEINPIA and also adjacentamino acids from the fifth extracellular loop of mpa. Preferably, thisfifth extracellular loop will be present in a polypeptide of theinvention in a suitable form and conformation for it to be recognised bythe immune system.

Preferably, the four polypeptides ahpC, gsd, p12 and mpa are providedtogether in a single fusion protein. The four polypeptide sequences insuch a fusion protein may be any of the polypeptides or variantsdescribed herein. The four polypeptides may be provided in any order inthe fusion protein. In one embodiment they are provided in the orderahpC-gsd-p12-mpa.

In one embodiment, the four polypeptides present in a fusion protein arethose given in SEQ ID Nos 4, 8, 12 and 16. For example, these fourproteins may be provided in a fusion protein in this order as shown inSEQ ID NO: 20.

In an alternative embodiment, the polypeptides may be present in two ormore separate polypeptide molecules, which may or may not be linked bynon-covalent linkages. For example, the four polypeptides may beprovided separately, or may be provided in two or three separate fusionprotein polypeptide molecules. For example, three of the polypeptidesmay be provided in a single polypeptide molecule and the fourth providedseparately, two may be provided in one molecule and the other twoprovided separately, or the four polypeptides may be provided in twopolypeptide molecules, each comprising two of the four polypeptides.

In a fusion protein of the invention, linker sequences may separate therequired polypeptide sequences and/or there may or may not be additionalsequences present at the N terminal or C terminal of the peptide.Typically the fusion protein comprises 1, 2, 3, or more such linkers.The linkers are typically 1, 2, 3, 4 or more amino acids in length. Thusin the peptide 1, 2, 3 or all of the polypeptide sequences may becontiguous with each other or may be separated from each other, forexample by such linkers.

A polypeptide of the invention may comprise further additionalsequences, for example those encoded by the polynucleotides and vectorsdescribed below. For example, it may comprise additional epitopes,therapeutic polypeptides, adjuvants or immunomodulatory molecules.

The polypeptide may comprise a leader sequence, i.e. a sequence at ornear the amino terminus of the polypeptide that functions in targetingor regulation of the polypeptide. For example a sequence may be includedin the polypeptide that targets it to particular tissues in the body, orwhich helps the processing or folding of the polypeptide uponexpression. Various such sequences are well known in the art and couldbe selected by the skilled reader depending upon, for example, thedesired properties and production method of the polypeptide. One exampleof such a leader is the ubiquitin leader sequence given in SEQ ID NO:17.

A polypeptide may further comprise a tag or label to identify or screenfor the polypeptide, or for expression of the polypeptide. Suitablelabels include radioisotopes such as ¹²⁵I, ³²P or ³⁵S, fluorescentlabels, enzyme labels, or other protein labels such as biotin. Suitabletags may be short amino acid sequences that can be identified by routinescreening methods. For example, a short amino acid sequence may beincluded that is recognised by a particular monoclonal antibody. Onesuch tag of the pK tag given in SEQ ID NO: 18.

In one embodiment, a polypeptide of the invention has the sequence givenin SEQ ID NO: 24. This is referred to herein as the Havilah polypeptidesequence and comprises the four modified polypeptides of SEQ ID Nos: 4,8, 12 and 16, and additional sequences such as a ubiquitin leadersequence and a pK tag.

Peptides of the invention, as defined herein, may be chemicallymodified, for example, post-translationally modified. For example theymay be glycosylated or comprise modified amino acid residues. They canbe in a variety of forms of polypeptide derivatives, including amidesand conjugates with polypeptides.

Chemically modified peptides also include those having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized side groups include those which have been derivatizedto form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxygroups, t-butyloxycarbonyl groups, chloroacetyl groups and formylgroups. Free carboxyl groups may be derivatized to form salts, methyland ethyl esters or other types of esters or hydrazides. Free hydroxylgroups may be derivatized to form O-acyl or O-alkyl derivatives. Theimidazole nitrogen of histidine may be derivatized to formN-im-benzylhistidine. Peptides may also be modified by phosphorylation,for example 3 amino phosphorylation and by glycosylation for examplemannosylation.

Also included as chemically modified peptides are those which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline or homoserine may be substituted for serine.

Polynucleotides

The invention also relates to polynucleotide constructs comprisingnucleic acid sequences which encode the four polypeptides or variantsthereof. For example, a single nucleic acid molecule may be providedwhich encodes an ahpC polypeptide, a gsd polypeptide, a p12 polypeptideand an mpa polypeptide. These four polypeptides may be encoded in anyorder in the nucleic acid molecule, but are preferably provided in theorder ahpC-gsd-p12-mpa. For example, a polynucleotide of the inventionmay encode any of the polypeptides or fusion proteins described above.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include a gene, a genefragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide of theinvention may be provided in isolated or purified form.

A nucleic acid sequence which “encodes” a selected polypeptide is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

In one embodiment, therefore, a polynucleotide of the inventioncomprises an ahpC gene sequence, a gsd gene sequence, a p12 genesequence and an mpa gene sequence. Suitable gene sequences are providedin SEQ ID Nos 1, 3, 5, 7, 9, 11, 13 and 15. A suitable ahpC, gsd, p12 ormpa sequence may alternatively be a variant of one of these specificsequences. For example, a variant may be a substitution, deletion oraddition variant of any of the above nucleic acid sequences. A variantof one of the four genes may comprise 1, 2, 3, 4, 5, up to 10, up to 20,up to 30, up to 40, up to 50, up to 75 or more nucleic acidsubstitutions and/or deletions from the sequences given in the sequencelisting.

Suitable variants may be at least 70% homologous to a MAP polynucleotideof the invention, preferably at least 80 or 90% and more preferably atleast 95%, 97% or 99% homologous thereto. Methods of measuring homologyare well known in the art and it will be understood by those of skill inthe art that in the present context, homology is calculated on the basisof nucleic acid identity. Such homology may exist over a region of atleast 15, preferably at least 30, for instance at least 40, 60, 100, 200or more contiguous nucleotides. Such homology may exist over the entirelength of the unmodified MAP polynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul,S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4,and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

The homologues typically hybridize with the relevant polynucleotide at alevel significantly above background. The signal level generated by theinteraction between the homologue and the polynucleotide is typically atleast 10 fold, preferably at least 100 fold, as intense as “backgroundhybridisation”. The intensity of interaction may be measured, forexample, by radiolabelling the probe, e.g. with ³²P. Selectivehybridisation is typically achieved using conditions of medium to highstringency, (for example, 0.03M sodium chloride and 0.003M sodiumcitrate at from about 50° C. to about 60° C.

Stringent hybridization conditions can include 50% formamide,5×Denhardt's Solution, 5×SSC, 0.1% SDS and 100 μg/1331 denatured salmonsperm DNA and the washing conditions can include 2×SSC, 0.1% SDS at 37°C. followed by 1×SSC, 0.1% SDS at 68° C. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra.

The homologue may differ from a sequence in the relevant polynucleotideby less than 3, 5, 10, 15, 20 or more mutations (each of which may be asubstitution, deletion or insertion). These mutations may be measuredover a region of at least 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specificsequences given in the sequence listing by virtue of the redundancy inthe genetic code. The DNA code has 4 primary nucleic acid residues (A,T, C and G) and uses these to “spell” three letter codons whichrepresent the amino acids the proteins encoded in an organism's genes.The linear sequence of codons along the DNA molecule is translated intothe linear sequence of amino acids in the protein(s) encoded by thosegenes. The code is highly degenerate, with 61 codons coding for the 20natural amino acids and 3 codons representing “stop” signals. Thus, mostamino acids are coded for by more than one codon—in fact several arecoded for by four or more different codons. A variant polynucleotide ofthe invention may therefore encode the same polypeptide sequence asanother polynucleotide of the invention, but may have a differentnucleic acid sequence due to the use of different codons to encode thesame amino acids.

In one embodiment the coding sequence of the polynucleotide constructmay be optimised to more closely resemble the codon usage of highlyexpressed genes in mammalian cells. Where more than one codon isavailable to code for a given amino acid, it has been observed that thecodon usage patterns of organisms are highly non-random. Differentspecies show a different bias in their codon selection and, furthermore,utilization of codons may be markedly different in a single speciesbetween genes which are expressed at high and low levels. This bias isdifferent in viruses, plants, bacteria and mammalian cells, and somespecies show a stronger bias away from a random codon selection thanothers.

For example, humans and other mammals are less strongly biased thancertain bacteria or viruses. For these reasons, it is possible that, forexample a mycobacterial gene expressed in mammalian cells will have aninappropriate distribution of codons for efficient expression. It isbelieved that the presence in a heterologous DNA sequence of clusters ofcodons which are rarely observed in the host in which expression is tooccur, is predictive of low heterologous expression levels in that host.

In the polynucleotide of the invention, the codon usage pattern maytherefore be altered from that found naturally in MAP to more closelyrepresent the codon bias of the target organism, e.g. a mammal,especially a human. The “codon usage coefficient” is a measure of howclosely the codon pattern of a given polynucleotide sequence resemblesthat of a target species. Codon frequencies can be derived fromliterature sources for the highly expressed genes of many species (seee.g. Nakamura et. al. Nucleic Acids Research 1996, 24:214-215). Thecodon frequencies for each of the 61 codons (expressed as the number ofoccurrences occurrence per 1000 codons of the selected class of genes)are normalised for each of the twenty natural amino acids, so that thevalue for the most frequently used codon for each amino acid is set to 1and the frequencies for the less common codons are scaled to lie betweenzero and 1. Thus each of the 61 codons is assigned a value of 1 or lowerfor the highly expressed genes of the target species. In order tocalculate a codon usage coefficient for a specific polynucleotide,relative to the highly expressed genes of that species, the scaled valuefor each codon of the specific polynucleotide are noted and thegeometric mean of all these values is taken (by dividing the sum of thenatural logs of these values by the total number of codons and take theanti-log). The coefficient will have a value between zero and 1 and thehigher the coefficient the more codons in the polynucleotide are“frequently used codons”. If a polynucleotide sequence has a codon usagecoefficient of 1, all of the codons are “most frequent” codons forhighly expressed genes of the target species.

According to the present invention, the codon usage pattern of thepolynucleotide of the invention will preferably exclude codons with arelative synonymous codon usage (RSCU) value of less than 0.2 in highlyexpressed genes of the target organism. A RSCU value is the observednumber of codons divided by the number expected if all codons for thatamino acid were used equally frequently. The polynucleotide of theinvention will generally have a codon usage coefficient for highlyexpressed human genes of greater than 0.3, preferably greater than 0.4,most preferably greater than 0.5. Codon usage tables for human can alsobe found in GenBank.

It can thus be seen that the particular polynucleotide sequence whichencodes a polypeptide of the invention may be altered to optimise thecodons based on the species to be treated. As an example of this, theMAP sequences given in SEQ ID Nos: 1, 5, 9 and 13 have been codonoptimised for human use in the polynucleotides of SEQ ID Nos: 3, 7, 11and 17. Such modifications may improve the ability of suchpolynucleotides to express their encoded proteins in a human cell.

As explained above in relation to polypeptides, the polynucleotides ofthe invention may also be modified to disable or remove potentialcross-reacting epitopes in the encoded polypeptide.

Polynucleotide “fragments” according to the invention may be made bytruncation, e.g. by removal of one or more nucleotides from one or bothends of a polynucleotide. Up to 10, up to 20, up to 30, up to 40, up to50, up to 75, up to 100, up to 200 or more amino acids may be removedfrom the 3′ and/or 5′ end of the polynucleotide in this way. Fragmentsmay also be generated by one or more internal deletions. For example, avariant of the invention may encode a polypeptide that consists of orcomprises two or more epitope regions from a full length polypeptide ofthe invention in the absence of non-epitope amino acids. Preferably afragment of an ahpC, gsd, p12 or mpa polynucleotide sequence comprisesat least one region encoding an epitope capable of inducing an immuneresponse against the unmodified MAP polypeptide. Such fragments may bederived from a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 or maybe derived from a variant polynucleotide as described herein. Preferablysuch fragments are between 24 and 500 residues in length, e.g. 24 to400, 24 to 300, 24 to 100, 100 to 200 or 200 to 400 residues.Alternatively, fragments of the invention may be longer sequences, forexample comprising at least 50%, at least 60%, at least 70%, at least80% or at least 90% of a full length polynucleotide of the invention.

A peptide of the invention may thus be produced from or delivered in theform of a polynucleotide which encodes, and is capable of expressing,it. Polynucleotides of the invention can be synthesised according tomethods well known in the art, as described by way of example inSambrook et al (1989, Molecular Cloning—a laboratory manual; Cold SpringHarbor Press). Substantially pure antigen preparations can be obtainedusing standard molecular biological tools. That is, polynucleotidesequences coding for the above-described moieties can be obtained usingrecombinant methods, such as by screening cDNA and genomic librariesfrom cells expressing an antigen, or by deriving the coding sequence fora polypeptide from a vector known to include the same. Furthermore, thedesired sequences can be isolated directly from cells and tissuescontaining the same, using standard techniques, such as phenolextraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA.Polynucleotide sequences can also be produced synthetically, rather thancloned.

Yet another convenient method for isolating specific nucleic acidmolecules is by the polymerase chain reaction (PCR). Mullis et al.(1987) Methods Enzymol. 155:335-350. This technique uses DNA polymerase,usually a thermostable DNA polymerase, to replicate a desired region ofDNA. The region of DNA to be replicated is identified byoligonucleotides of specified sequence complementary to opposite endsand opposite strands of the desired DNA to prime the replicationreaction. The product of the first round of replication is itself atemplate for subsequent replication, thus repeated successive cycles ofreplication result in geometric amplification of the DNA fragmentdelimited by the primer pair used.

Once the sequences have been obtained, they may be linked together toprovide a nucleic acid molecule using standard cloning or molecularbiology techniques. Alternatively, the sequences can be producedsynthetically, rather than cloned. The nucleotide sequence can bedesigned with the appropriate codons for the particular amino acidsequence desired. As explained herein, one will generally selectpreferred codons for the intended host in which the sequence will beexpressed. The complete sequence can then be assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence.

Vectors

The nucleic acid molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the polypeptide of the invention in vivo in a targeted subjectspecies. These expression cassettes, in turn, are typically providedwithin vectors (e.g., plasmids or recombinant viral vectors) which aresuitable for use as reagents for nucleic acid immunization. Such anexpression cassette may be administered directly to a host subject.Alternatively, a vector comprising a polynucleotide of the invention maybe administered to a host subject. Preferably the polynucleotide isprepared and/or administered using a genetic vector. A suitable vectormay be any vector which is capable of carrying a sufficient amount ofgenetic information, and allowing expression of a polypeptide of theinvention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which axe positioned in the correct orientation,in order to allow for expression of a peptide of the invention. Othersuitable vectors would be apparent to persons skilled in the art. By wayof further example in this regard we refer to Sambrook et al.

Thus, a polypeptide of the invention may be provided by delivering sucha vector to a cell and allowing transcription from the vector to occur.Preferably, a polynucleotide of the invention or for use in theinvention in a vector is operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by thehost cell, i.e. the vector is an expression vector.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given regulatory sequence, such as a promoter,operably linked to a nucleic acid sequence is capable of effecting theexpression of that sequence when the proper enzymes are present. Thepromoter need not be contiguous with the sequence, so long as itfunctions to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween the promoter sequence and the nucleic acid sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

A number of expression systems have been described in the art, each ofwhich typically consists of a vector containing a gene or nucleotidesequence of interest operably linked to expression control sequences.These control sequences include transcriptional promoter sequences andtranscriptional start and termination sequences. The vectors of theinvention may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter. A“plasmid” is a vector in the form of an extrachromosomal geneticelement. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a resistance gene for a fungal vector. Vectors may be used invitro, for example for the production of DNA or RNA or used to transfector transform a host cell, for example, a mammalian host cell. Thevectors may also be adapted to be used in vivo, for example to allow invivo expression of the polypeptide.

A “promoter” is a nucleotide sequence which initiates and regulatestranscription of a polypeptide-encoding polynucleotide. Promoters caninclude inducible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), repressible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris repressed by an analyte, cofactor, regulatory protein, etc.), andconstitutive promoters. It is intended that the term “promoter” or“control element” includes full-length promoter regions and functional(e.g., controls transcription or translation) segments of these regions.

Promoters and other expression regulation signals may be selected to becompatible with the host cell for which expression is designed. Forexample, yeast promoters include S. cerevisiae GAL4 and ADH promoters,S. pombe nmt1 and adh promoter. Mammalian promoters, such as β-actinpromoters, may be used. Tissue-specific promoters are especiallypreferred. Mammalian promoters include the metallothionein promoterwhich can be induced in response to heavy metals such as cadmium.

In one embodiment a viral promoter is used to drive expression from thepolynucleotide. Typical viral promoters for mammalian cell expressioninclude the SV40 large T antigen promoter, adenovirus promoters, theMoloney murine leukaemia virus long terminal repeat (MMLV LTR), themouse mammary tumor virus LTR promoter, the rous sarcoma virus (RSV) LTRpromoter, the SV40 early promoter, the human cytomegalovirus (CMV) IEpromoter, adenovirus, including the adenovirus major late promoter (AdMLP), HSV promoters (such as the HSV IE promoters), or HPV promoters,particularly the HPV upstream regulatory region (URR). All thesepromoters are readily available in the art.

In one embodiment, the promoter is a Cytomegalovirus (CMV) promoter. Apreferred promoter element is the CMV immediate early (IE) promoterdevoid of intron A, but including exon. 1. Thus the expression from thepolynucleotide may be under the control of hCMV IE early promoter.Expression vectors using the hCMV immediate early promoter include forexample, pWRG7128, and pBC12/CMV and pJW4303. A hCMV immediate earlypromoter sequence can be obtained using known methods. A native hCMVimmediate early promoter can be isolated directly from a sample of thevirus, using standard techniques. U.S. Pat. No. 5,385,839, for example,describes the cloning of a hCMV promoter region. The sequence of a hCMVimmediate early promoter is available at Genbank #M60321 (hCMV Townestrain) and X17403 (hCMV Ad169 strain). A native sequence couldtherefore be isolated by PCR using PCR primers based on the knownsequence. See e.g Sambrook et al, supra, for a description of techniquesused to obtain and isolate DNA. A suitable hCMV promoter sequence couldalso be isolated from an existing plasmid vector. Promoter sequences canalso be produced synthetically.

A polynucleotide, expression cassette or vector of the invention maycomprise an untranslated leader sequence. In general the untranslatedleader sequence has a length of from about 10 to about 200 nucleotides,for example from about 15 to 150 nucleotides, preferably 15 to about 130nucleotides. Leader sequences comprising, for example, 15, 50, 75 or 100nucleotides may be used. Generally a functional untranslated leadersequence is one which is able to provide a translational start site forexpression of a coding sequence in operable linkage with the leadersequence.

Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence. Introns, containing splice donor and acceptorsites, may also be designed into the expression cassette or vector.

Expression systems often include transcriptional modulator elements,referred to as “enhancers”. Enhancers are broadly defined as acis-acting agent, which when operably linked to a promoter/genesequence, will increase transcription of that gene sequence. Enhancerscan function from positions that are much further away from a sequenceof interest than other expression control elements (e.g. promoters), andmay operate when positioned in either orientation relative to thesequence of interest. Enhancers have been identified from a number ofviral sources, including polyoma virus, BK virus, cytomegalovirus (CMV),adenovirus, simian virus 40 (SV40), Moloney sarcoma virus, bovinepapilloma virus and Rous sarcoma virus. Examples of suitable enhancersinclude the SV40 early gene enhancer, the enhancer/promoter derived fromthe long terminal repeat (LTR) of the Rous Sarcoma Virus, and elementsderived from human or murine CMV, for example, elements included in theCMV intron A sequence.

A polynucleotide, expression cassette or vector according to the presentinvention may additionally comprise a signal peptide sequence. Thesignal peptide sequence is generally inserted in operable linkage withthe promoter such that the signal peptide is expressed and facilitatessecretion of a polypeptide encoded by coding sequence also in operablelinkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 aminoacids for example 15 to 20 amino acids. Often the amino acids arepredominantly hydrophobic. In a typical situation, a signal peptidetargets a growing polypeptide chain bearing the signal peptide to theendoplasmic reticulum of the expressing cell. The signal peptide iscleaved off in the endoplasmic reticulum, allowing for secretion of thepolypeptide via the Golgi apparatus.

Nucleic acids encoding for polypeptides known to display antiviral orantibacterial activity, immunomodulatory molecules such as cytokines(e.g. TNF-alpha, interferons such as IL-6, and IL-2, interferons, colonystimulating factors such as GM-CSF), adjuvants and co-stimulatory andaccessory molecules (B7-1, B7-2) may be included in a polynucleotide,expression cassette or vector of the invention. Alternatively, suchpolypeptides may be provided separately, for example in a formulationcomprising a molecule of the invention, or may be administeredsimultaneously, sequentially or separately with a composition of theinvention. Concurrent provision of an immunomodulatory molecule and apolypeptide of the invention at a site in vivo may enhance thegeneration of specific effectors which may help to enhance the immuneresponse. The degree of enhancement of the immune response may bedependent upon the specific immunostimulatory molecules and/or adjuvantsused because different immunostimulatory molecules may elicit differentmechanisms for enhancing and/or modulating the immune response. By wayof example, the different effector mechanisms/immunomodulatory moleculesinclude but are not limited to augmentation of help signal (IL-2),recruitment of professional APC (GM-CSF), increase in T cell frequency(IL-2), effect on antigen processing pathway and MHC expression(IFN-gamma and TNF-alpha) and diversion of immune response away from theTh1 response and towards a Th2 response. Unmethylated CpG containingoligonucleotides are also preferential inducers of a Th1 response andare suitable for use in the present invention.

In some embodiments, the polynucleotide, expression cassette or vectorwill encode an adjuvant, or an adjuvant will otherwise be provided. Asused herein, the term “adjuvant” refers to any material or compositioncapable of specifically or non-specifically altering, enhancing,directing, redirecting, potentiating or initiating an antigen-specificimmune response.

A suitable adjuvant may be an ADP-ribosylating bacterial toxin. Theseinclude diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT),the E. coli heat labile toxins (LT1 and LT2), Pseudomonas endotoxin A,Pseudomonas exotoxin S, B. cereus exoenzyme, B. sphaericus toxin, C.botulinum C2 and C3 toxins, C. limosum exoenzyme, as well as toxins fromC. perfringens, C. spiriforma and C. difficile and Staphylococcus aureusEDIN. Most ADP-ribosylating bacterial toxins contain A and B subunits.

Polynucleotides of interest may be used in vitro or in vivo in theproduction of a peptide of the invention. Such polynucleotides may beadministered or used in the manufacture of a medicament for thetreatment of Crohn's disease or another disease or conditioncharacterised by the expression of MAP.

Gene therapy and nucleic acid immunization are approaches which providefor the introduction of a nucleic acid molecule encoding one or moreselected antigens into a host cell for the in vivo expression of theantigen or antigens. Methods for gene delivery are known in the art.See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859 and 5,589,466. Thenucleic acid molecule can be introduced directly into the recipientsubject, such as by standard intramuscular or intradermal injection;transdermal particle delivery; inhalation; topically, or by oral,intranasal or mucosal modes of administration. The moleculealternatively can be introduced ex vivo into cells which have beenremoved from a subject. In this latter case, cells containing thenucleic acid molecule of interest are re-introduced into the subjectsuch that an immune response can be mounted against the antigen encodedby the nucleic acid molecule. The nucleic acid molecules used in suchimmunization are generally referred to herein as “nucleic acidvaccines.”

Each of these delivery techniques requires efficient expression of thenucleic acid in the transfected cell, to provide a sufficient amount ofthe therapeutic or antigenic gene product. Several factors are known toaffect the levels of expression obtained, including transfectionefficiency, and the efficiency with which the gene or sequence ofinterest is transcribed and the mRNA translated.

The agent produced by a host cell may be secreted or may be containedintracellularly depending on the polynucleotide and/or the vector used.As will be understood by those of skill in the art, expression vectorscontaining the polynucleotides of the invention can be designed withsignal sequences which direct secretion of the polypeptide expressedfrom the vector through a particular prokaryotic or eukaryotic cellmembrane.

The vectors and expression cassettes of the present invention may beadministered directly as “a naked nucleic acid construct”, preferablyfurther comprising flanking sequences homologous to the host cellgenome. As used herein, the term “naked DNA” refers to a vector such asa plasmid comprising a polynucleotide of the present invention togetherwith a short promoter region to control its production. It is called“naked” DNA because the vectors are not carried in any delivery vehicle.When such a vector enters a host cell, such as a eukaryotic cell, theproteins it encodes are transcribed and translated within the cell.

The vector of the invention may thus be a plasmid vector, that is, anautonomously replicating, extrachromosomal circular or linear DNAmolecule. The plasmid may include additional elements, such as an originof replication, or selector genes. Such elements are known in the artand can be included using standard techniques. Numerous suitableexpression plasmids are known in the art. For example, one suitableplasmid is pSG2. This plasmid was originally isolated from Streptomycesghanaensis. The length of 13.8 kb, single restriction sites for HindIII,EcoRV and PvuII and the possibility of deleting non-essential regions ofthe plasmid make pSG2 a suitable basic replicon for vector development.

Alternatively, the vectors of the present invention may be introducedinto suitable host cells using a variety of viral techniques which areknown in the art, such as for example infection with recombinant viralvectors such as retroviruses, herpes simplex viruses and adenoviruses.

In one embodiment, the vector itself may be a recombinant viral vector.Suitable recombinant viral vectors include but are not limited toadenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virusvectors, a retroviral vector, lentiviral vectors, baculoviral vectors,pox viral vectors or parvovirus vectors. In the case of viral vectors,administration of the polynucleotide is mediated by viral infection of atarget cell.

A number of viral based systems have been developed for transfectingmammalian cells.

For example, a selected recombinant nucleic acid molecule can beinserted into a vector and packaged as retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to cells of the subject either in vivo or ex vivo.Retroviral vectors may be based upon the Moloney murine leukaemia virus(Mo-MLV). In a retroviral vector, one or more of the viral genes (gag,pol & env) are generally replaced with the gene of interest.

A number of adenovirus vectors are known. Adenovirus subgroup Cserotypes 2 and 5 are commonly used as vectors. The wild type adenovirusgenome is approximately 35 kb of which up to 30 kb can be replaced withforeign DNA. There are four early transcriptional units (E1, E2, E3 &E4), which have regulatory functions, & a late transcript, which codesfor structural proteins. Adenovirus vectors may have the E1 and/or E3gene inactivated. The missing gene(s) may then be supplied in transeither by a helper virus, plasmid or integrated into a helper cellgenome. Adenovirus vectors may use an E2a temperature sensitive mutantor an E4 deletion. Minimal adenovirus vectors may contain only theinverted terminal repeats (ITRs) & a packaging sequence around thetransgene, all the necessary viral genes being provided in trans by ahelper virus. Suitable adenoviral vectors thus include Ad5 vectors andsimian adenovirus vectors.

Viral vectors may also be derived from the pox family of viruses,including vaccinia viruses and avian poxvirus such as fowlpox vaccines.For example, modified vaccinia virus Ankara (MVA) is a strain ofvaccinia virus which does not replicate in most cell types, includingnormal human tissues. A recombinant MVA vector may therefore be used todeliver the polypeptide of the invention.

Addition types of virus such as adeno-associated virus (AAV) and herpessimplex virus (HSV) may also be used to develop suitable vector systems.

As an alternative to viral vectors, liposomal preparations canalternatively be used to deliver the nucleic acid molecules of theinvention. Useful liposomal preparations include cationic (positivelycharged), anionic (negatively charged) and neutral preparations, withcationic liposomes particularly preferred. Cationic liposomes maymediate intracellular delivery of plasmid DNA and mRNA.

As another alternative to viral vector systems, the nucleic acidmolecules of the present invention may be encapsulated, adsorbed to, orassociated with, particulate carriers. Suitable particulate carriersinclude those derived from polymethyl methacrylate polymers, as well asPLG microparticles derived from poly(lactides) andpoly(lactide-co-glycolides). Other particulate systems and polymers canalso be used, for example, polymers such as polylysine, polyarginine,polyornithine, spermine, spermidine, as well as conjugates of thesemolecules.

In one embodiment, the vector may be a targeted vector, that is a vectorwhose ability to infect or transfect or transduce a cell or to beexpressed in a host and/or target cell is restricted to certain celltypes within the host subject, usually cells having a common or similarphenotype.

Preferably, a vector of the invention encodes an ahpC, a gsd, a p12 andan mpa polypeptide. As explained above, these four polypeptides may beexpressed together as a single fusion protein molecule, or may beexpressed in two or more separate polypeptides, each comprising one ormore of the individual components. The vector of the invention may thuscomprise a single expression cassette, from which a single polypeptidesequence can be expressed. Alternatively, a vector of the invention maycomprise two or more expression cassettes each capable of expressing adifferent polypeptide, such that the vector as a whole is capable ofexpressing all four required polypeptides. In one embodiment, a vectorof the invention may express all four required polypeptides separatelyas separate polypeptide molecules. Where the polypeptides are expressedfrom more than one locus in the vector, or are expressed as multipleseparate molecules, the expression of the multiple sequences ispreferably coordinated such that all four polypeptides are expressedtogether. For example, the same or similar promoters may be used tocontrol expression of the various components. Inducible promoters may beused so that expression of the various polypeptide components can becoordinated.

Cell Lines

The invention also includes cells that have been modified to express apeptide of the invention. Such cells include transient, or preferablystable higher eukaryotic cell lines, such as mammalian cells or insectcells, lower eukaryotic cells, such as yeast or prokaryotic cells suchas bacterial cells. Particular examples of cells which may be modifiedby insertion of vectors or expression cassettes encoding for a peptideof the invention include mammalian HEK293T, CHO, HeLa and COS cells.Preferably the cell line selected will be one which is not only stable,but also allows for mature glycosylation and cell surface expression ofa polypeptide. Expression may be achieved in transformed oocytes. Asuitable peptide may be expressed in cells of a transgenic non-humananimal, preferably a mouse. A transgenic non-human animal expressing apeptide of the invention is included within the scope of the invention.A peptide of the invention may also be expressed in Xenopus laevisoocytes or melanophores.

Such cell lines of the invention may be cultured using routine methodsto produce a polypeptide of the invention, or may be usedtherapeutically or prophylactically to deliver polypeptides of theinvention to a subject. For example, cell lines capable of secreting apolypeptide of the invention may be administered to a subject.Alternatively, polynucleotides, expression cassettes or vectors of theinvention may be administered to a cell from a subject ex vivo and thecell then returned to the body of the subject.

For example, methods for the ex vivo delivery and reimplantation oftransformed cells into a subject are known (e.g., dextran-mediatedtransfection, calcium phosphate precipitation, electroporation, anddirect microinjection into nuclei).

Antibodies

The present invention also extends to antibodies (monoclonal orpolyclonal) and their antigen-binding fragments (e.g. F(ab)2, Fab and Fvfragments i.e. fragments of the “variable” region of the antibody, whichcomprises the antigen binding site) directed to peptides as definedhereinbefore, i.e. which bind to epitopes present on the peptides andthus bind selectively and specifically to such peptides, and which maybe used in the methods of the invention. For example, a polyclonalantibody may be produced which has a broad spectrum effect against avariety of epitopes on a polypeptide of the invention.

Pharmaceutical Compositions

Formulation of a composition comprising a molecule of the invention,such as a polynucleotide, expression cassette, vector, polypeptide, cellor antibody as described above, can be carried out using standardpharmaceutical formulation chemistries and methodologies all of whichare readily available to the reasonably skilled artisan. For example,compositions containing one or more molecules of the invention can becombined with one or more pharmaceutically acceptable excipients orvehicles. Auxiliary substances, such as wetting or emulsifying agents,pH buffering substances and the like, may be present in the excipient orvehicle. These excipients, vehicles and auxiliary substances aregenerally pharmaceutical agents that do not induce an immune response inthe individual receiving the composition, and which may be administeredwithout undue toxicity. Pharmaceutically acceptable excipients include,but are not limited to, liquids such as water, saline,polyethyleneglycol, hyaluronic acid, glycerol and ethanol.Pharmaceutically acceptable salts can also be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients, vehicles andauxiliary substances is available in Remington's Pharmaceutical Sciences(Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitablefor bolus administration or for continuous administration. Injectablecompositions may be prepared, packaged, or sold in unit dosage form,such as in ampoules or in multi-dose containers containing apreservative. Compositions include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchcompositions may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a composition for parenteraladministration, the active ingredient is provided in dry (for e.g., apowder or granules) form for reconstitution with a suitable vehicle(e.g., sterile pyrogen-free water) prior to parenteral administration ofthe reconstituted composition. The pharmaceutical compositions may beprepared, packaged, or sold in the form of a sterile injectable aqueousor oily suspension or solution. This suspension or solution may beformulated according to the known art, and may comprise, in addition tothe active ingredient, additional ingredients such as the dispersingagents, wetting agents, or suspending agents described herein. Suchsterile injectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides.

Other parentally-administrable compositions which are useful includethose which comprise the active ingredient in microcrystalline form, ina liposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

Certain facilitators of nucleic acid uptake and/or expression(“transfection facilitating agents”) can also be included in thecompositions, for example, facilitators such as bupivacaine, cardiotoxinand sucrose, and transfection facilitating vehicles such as liposomal orlipid preparations that are routinely used to deliver nucleic acidmolecules. Anionic and neutral liposomes are widely available and wellknown for delivering nucleic acid molecules (see, e.g., Liposomes: APractical Approach, (1990) RPC New Ed., IRL Press). Cationic lipidpreparations are also well known vehicles for use in delivery of nucleicacid molecules. Suitable lipid preparations include DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride),available under the tradename Lipofectin™, and DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio)propane), see, e.g., Feigner etal. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416; Malone et al. (1989)Proc. Natl. Acad. Sci. USA 86:6077-6081; U.S. Pat. Nos. 5,283,185 and5,527,928, and International Publication Nos WO 90/11092, WO 91/15501and WO 95/26356. These cationic lipids may preferably be used inassociation with a neutral lipid, for example DOPE (dioleylphosphatidylethanolamine). Still further transfection-facilitatingcompositions that can be added to the above lipid or liposomepreparations include spermine derivatives (see, e.g., InternationalPublication No. WO 93/18759) and membrane-permeabilizing compounds suchas GALA, Gramicidine S and cationic bile salts (see, e.g., InternationalPublication No. WO 93/19768).

Alternatively, the nucleic acid molecules of the present invention maybe encapsulated, adsorbed to, or associated with, particulate carriers.Suitable particulate carriers include those derived from polymethylmethacrylate polymers, as well as PLG microparticles derived frompoly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery etal. (1993) Pharm. Res. 10:362-368. Other particulate systems andpolymers can also be used, for example, polymers such as polylysine,polyarginine, polyornithine, spermine, spermidine, as well as conjugatesof these molecules.

The formulated compositions will include an amount of the molecule (e.g.vector) of interest which is sufficient to mount an immunologicalresponse. An appropriate effective amount can be readily determined byone of skill in the art. Such an amount will fall in a relatively broadrange that can be determined through routine trials. The compositionsmay contain from about 0.1% to about 99.9% of the vector and can beadministered directly to the subject or, alternatively, delivered exvivo, to cells derived from the subject, using methods known to thoseskilled in the art.

Therapeutic Methods

The present invention relates to immunogenic molecules which areintended to direct an immune response against MAP. The compositions ofthe invention can thus be used in the treatment or prevention ofinfection by MAP, or in the treatment or prevention of any disease,condition or symptom which is associated with MAP infection, that is anydisease condition or symptom which is a direct or indirect result of MAPinfection, or which results from a disease or condition to which thepresence of MAP contributes. MAP is known to be linked to numerousspecific medical conditions, such as chronic inflammation of theintestine, including inflammatory bowel disease and as well as IrritableBowel Syndrome. For example, MAP infection can cause chronic enteritis,such as Johne's disease (paratuberculosis) in livestock and Crohn'sdisease and Irritable Bowel Syndrome in humans. The compositions of theinvention may therefore be used in the prevention or treatment of any ofthese specific conditions.

Accordingly, the present invention relates to a polypeptide,polynucleotide, expression cassette, vector, cell, antibody orcomposition of the invention for use in a method of therapy, inparticular in a method or treating or preventing a disease, disorder orsymptoms associated with or caused by a MAP infection. These moleculesof the invention may thus also be used in the manufacture of amedicament for treating or preventing such a disease, disorder orcondition. In particular, the molecules of the invention are proposedfor the treatment or prevention of a chronic inflammation of theintestine, preferably in a mammal such as a human, cow, sheep or goat.The invention thus also provides a method of treating or preventing anysuch disease, disorder or symptom comprising administering to a subjectin need thereof a polypeptide, polynucleotide, expression cassette,vector, cell, antibody or composition of the invention.

The present invention is broadly applicable to vaccination methods andis relevant to the development of prophylactic and/or therapeuticvaccines (including immunotherapeutic vaccines). It is to be appreciatedthat all references herein to treatment include curative, palliative andprophylactic treatment.

According to the present invention, a polynucleotide, vector,polypeptide or other molecule of the invention may be employed alone aspart of a composition, such as but not limited to a pharmaceuticalcomposition or a vaccine composition or an immunotherapeutic compositionto prevent and/or treat a condition associated with MAP infection. Theadministration of the composition may be for either “prophylactic” or“therapeutic” purpose. As used herein, the term “therapeutic” or“treatment” includes any of following: the prevention of infection orreinfection; the reduction or elimination of symptoms; and the reductionor complete elimination of a pathogen. Treatment may be effectedprophylactically (prior to infection) or therapeutically (followinginfection).

Prophylaxis or therapy includes but is not limited to eliciting aneffective immune response to a polypeptide of the invention and/oralleviating, reducing, curing or at least partially arresting symptomsand/or complications resulting from or associated with a MAP infection.When provided prophylactically, the composition of the present inventionis typically provided in advance of any symptom. The prophylacticadministration of the composition of the present invention is to preventor ameliorate any subsequent infection or disease. When providedtherapeutically, the composition of the present invention is typicallyprovided at or shortly after the onset of a symptom of infection ordisease. Thus the composition of the present invention may be providedeither prior to the anticipated exposure to MAP or onset of theassociated disease state or after the initiation of an infection ordisease.

Subject to be Treated

The present invention relates in particular to the treatment orprevention of diseases or other conditions which are associated withinfection by MAP. These treatments may be used on any animal which issusceptible to infection by MAP.

The subject to be treated may be any member of the subphylum cordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The terms do not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The methods described herein are intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly. If a mammal, the subject will preferablybe a human, but may also be a domestic livestock, laboratory subject orpet animal.

The subject to be treated may thus be any vertebrate that is susceptibleto infection by MAP. Numerous animals have been shown in the art to becapable of such infection, including livestock such as cattle, goat andsheep, primates such as macaques and humans, other mammals includingalpaca, antelope, ass, elk, horses, deer, dogs, gerbils and rabbits, andbirds including the chicken. The compositions of the present inventionmay thus be used in the treatment of any such species.

Combined Therapy

In one instance, a molecule of the invention may be used in combinationwith another molecule, such as another polynucleotide, vector orpolypeptide, preferably another therapeutic agent. The therapeutic agentmay be, for example an agent which has activity against MAP, or an agentused in the treatment of a condition which is associated with MAPinfection. The molecule of the invention is preferably administered inan amount which is sufficient to augment the anti-MAP effects of theother therapeutic agent or vice versa. Numerous other agents may be usedin the treatment of MAP or conditions which are associated with MAPinfection. These include the rifamycins such as rifabutin and rifaximin,clarithromycin and other macrolides. Various anti tuberculosis drugs mayalso be used.

The other therapeutic agent may be an agent which potentiates theeffects of the molecule of the invention. For example, the other agentmay be an immunomodulatory molecule or an adjuvant which enhances theimmune response to the polypeptide of the invention. Alternatively, theother molecule may increase the susceptibility of MAP present in thesubject to attack, such as attack from the immune system.

In one embodiment, therefore, a molecule of the invention is used fortherapy in combination with one or more other therapeutic agents.

The two molecules may be administered separately, simultaneously orsequentially. The two may be administered in the same or differentcompositions. Accordingly, in a method of the invention, the subject mayalso be treated with a further therapeutic agent.

A composition may therefore be formulated which comprises a molecule ofthe invention and also one or more other therapeutic molecules. Forexample, a vector of the invention may be formulated with another vectorwhich encodes one or more other antigens or therapeutic molecules. Avector of the invention may alternatively be formulated with one or moretherapeutic proteins.

A composition of the invention may alternatively by used simultaneously,sequentially or separately with one or more other therapeutic conditionsas part of a combined treatment. Thus the invention also provides theuse of a molecule, such as a polypeptide, polynucleotide, vector or hostcell of the invention, in the manufacture of one or more medicament(s)for the treatment or prevention of MAP infection or a disease, conditionor symptom associated with MAP infection as described herein.

Delivery Methods

Once formulated the compositions can be delivered to a subject in vivousing a variety of known routes and techniques. For example, acomposition can be provided as an injectable solution, suspension oremulsion and administered via parenteral, subcutaneous, epidermal,intradermal, intramuscular, intraarterial, intraperitoneal, intravenousinjection using a conventional needle and syringe, or using a liquid jetinjection system. Compositions can also be administered topically toskin or mucosal tissue, such as nasally, intratracheally, intestinal,rectally or vaginally, or provided as a finely divided spray suitablefor respiratory or pulmonary administration. Other modes ofadministration include oral administration, suppositories, and active orpassive transdermal delivery techniques. Particularly in relation to thepresent invention, compositions may be administered directly to thegastrointestinal tract.

Alternatively, the compositions can be administered ex vivo, for exampledelivery and reimplantation of transformed cells into a subject areknown (e.g., dextran-mediated transfection, calcium phosphateprecipitation, electroporation, and direct microinjection into nuclei).

Delivery Regimes

The compositions are administered to a subject in an amount that iscompatible with the dosage formulation and that will be prophylacticallyand/or therapeutically effective. An appropriate effective amount willfall in a relatively broad range but can be readily determined by one ofskill in the art by routine trials. The “Physicians Desk Reference” and“Goodman and Gilman's The Pharmacological Basis of Therapeutics” areuseful for the purpose of determining the amount needed.

As used herein, the term “prophylactically or therapeutically effectivedose” means a dose in an amount sufficient to elicit an immune responseto one or more epitopes of a polypeptide of the invention and/or toalleviate, reduce, cure or at least partially arrest symptoms and/orcomplications from a disease, such as an inflammatory bowel disorder,which is associated with a MAP infection.

Prophylaxis or therapy can be accomplished by a single directadministration at a single time point or by multiple administrations,optionally at multiple time points. Administration can also be deliveredto a single or to multiple sites. Those skilled in the art can adjustthe dosage and concentration to suit the particular route of delivery.In one embodiment, a single dose is administered on a single occasion.In an alternative embodiment, a number of doses are administered to asubject on the same occasion but, for example, at different sites. In afurther embodiment, multiple doses are administered on multipleoccasions. Such multiple doses may be administered in batches, i.e. withmultiple administrations at different sites on the same occasion, or maybe administered individually, with one administration on each ofmultiple occasions (optionally at multiple sites). Any combination ofsuch administration regimes may be used.

In one embodiment, different compositions of the invention may beadministered at different sites or on different occasions as part of thesame treatment regime. It is known that improved immune responses may begenerated to an antigen by varying the vectors used to deliver theantigen. There is evidence that in some instances antibody and/orcellular immune responses may be improved by using two different vectorsadministered sequentially as a “prime” and a “boost”.

For example, the same polynucleotide of the invention may beadministered as a “prime” in one composition, and then subsequentlyadministered as a “boost” in a different composition. The two vaccinecompositions may differ in the choice of vector comprising thepolynucleotide. For example, two or more of different vectors eachselected from plasmid vectors, poxvirus vectors, adenovirus vectors orother vectors as described herein may be administered sequentially.

In one embodiment, a “prime” is effected by administering apolynucleotide of the invention, such as the Havilah polynucleotide ofSEQ ID NO: 23, in a plasmid vector such as pSG2. A “boost” is theneffected at a later time using a polynucleotide of the invention, suchas the Havilah polynucleotide of SEQ ID NO: 23 in a poxvirus vector suchas MVA.

In an alternative embodiment a “prime” is effected by administering apolynucleotide of the invention, such as the Havilah polynucleotide ofSEQ ID NO: 23, in an adenovirus vector such as Ad5. A “boost” is theneffected at a later time using a polynucleotide of the invention, suchas the Havilah polynucleotide of SEQ ID NO: 23 in a poxvirus vector suchas MVA.

In such a prime-boost protocol, one or more administrations of the primeand/or the boost may be performed. For example, the prime and/or booststep may be achieved using a single administration or using two or moreadministrations at different sites and/or on different occasions. In oneembodiment, two administrations on different occasions are given for theprime step and a single administration on a later occasion is given forthe boost step.

Different administrations may be performed on the same occasion, on thesame day, one, two, three, four, five or six days apart, one, two,three, four or more weeks apart. Preferably, administrations are 1 to 5weeks apart, more preferably 2 to 4 weeks apart, such as 2 weeks, 3weeks or 4 weeks apart. The schedule and timing of such multipleadministrations can be optimised for a particular composition orcompositions by one of skill in the art by routine trials.

Dosages for administration will depend upon a number of factorsincluding the nature of the composition, the route of administration andthe schedule and timing of the administration regime. Suitable doses ofa molecule of the invention may be in the order of up to 15 μg, up to 20μg, up to 25 μg, up to 30 μg, up to 50 μg, up to 100 μg, up to 500 μg ormore per administration. For some molecules of the invention, such asplasmids, the dose used may be higher, for example, up to 1 mg, up to 2mg, up to 3 mg, up to 4 mg, up to 5 mg or higher. Such doses may beprovided in a liquid formulation, at a concentration suitable to allowan appropriate volume for administration by the selected route. In thecase of a viral vector, a dose of about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ or morepfu may be given per administration. For example, a dose of 10⁹ pfu or25 μg of a vector of the invention may be administered in a 50 μl doseat multiple sites and/or on multiple occasions.

Kits

The invention also relates to a combination of components describedherein suitable for use in a treatment of the invention which arepackaged in the form of a kit in a container. Such kits may comprise aseries of components to allow for a treatment of the invention. Forexample, a kit may comprise two or more different vectors of theinvention, or one or more vectors of the invention and one or moreadditional therapeutic agents suitable for simultaneous administration,or for sequential or separate administration such as using a prime andboost protocol. The kit may optionally contain other suitablereagent(s), control(s) or instructions and the like.

EXAMPLES 1. Production of Havilah (HAV) Construct and RecombinantVectors Expressing HAV

A targeted bioinformatic analysis of the MAP genome was carried out andtwo secreted and two membrane bound components each related to thepathogenic phenotype were selected. These are AhpC, gsd, p12 and mpa.Three of these 4 components are ‘seen’ by antibody in sera from Crohn'sdisease patients as well as by sera from MAP infected C57/BL6 mice.Detailed epitope scans using the available databases revealed multiplepredicted human class I and class II epitopes in the selected vaccinecomponents. The peptides comprising these epitopes, when matched againstthe available human genome sequence, did not reveal any potential crossreacting antigens as potential targets for autoimmunity.

A construct consisting of a fusion of these four antigens was assembledfrom 40 mer oligonucleotide precursors synthesized with optimalmammalian codon usage. Functional domains including potentialcross-reacting human epitopes, lipid acylation sites and hydrophobictransmembrane regions were excluded. A monoclonal antibody recognitionpeptide was added to the C-terminus and a short human ubiquitin leadersequence to the N-terminus. This construct is referred to herein asHavilah and has the nucleic acid sequence given in SEQ ID NO: 23.

The Havilah (HAV) construct was cloned into the pSG2 expression vectorand inserted by homologous recombination into the Modified VacciniaAnkara (MVA) vector pMVA-GFP2 which carried a fluorescent marker.pMVA-GFP2 was used to transform MVA carrying a red marker at the targetsite for insertion, so that successful recombinants changed from red togreen enabling them to be isolated using a fluorescence activated cellsorter. HAV was also inserted into the vector comprising replicationdefective human adenovirus 5. The rec.pSG2.HAV plasmid, rec.MVA.HAV, andrec.Ad5.hav were all shown to express the predicted 95 kDa HAV encodedpolyprotein. The successful rec.pSG2.HAV, rec.MVA.HAV and rec.Ad5.HAVand corresponding control vectors were prepared in bulk, purified andstored ready for testing in vivo, E. coli were transformed with theindividual components AhpC and mpa representing the upstream anddownstream ends of HAV and the His-tagged recombinant proteins werepurified. Libraries of synthetic 15 residue peptides spanning the entireamino acid sequences of Havilah were obtained (FIG. 4). The recombinantproteins and pools of the synthetic peptides were used to develop theELISPOT and ELISA assays required to monitor the immune responses to thevaccine.

2. Safety and Immunogenicity of Vaccination Using pSG2.HAV then MVA.HAV

Two groups of 6 naïve 5 week old C57/BL6 female mice were isolatormaintained. Their physical condition was monitored daily and bodyweights recorded twice weekly and at the end of the study. After aninitial settle-in period of 7 days control Group 1 mice were primevaccinated with 25 μg of pSG2 expression plasmid in 50 μl sterilebuffered saline i.m. into each thigh. The experimental group receivedthe same vaccination using recombinant pSG2.Hav plasmid expressing theHavilah construct. Ten days later Group 1 mice were boost vaccinatedi.v. with 10⁶ pfu Modified Vaccinia Ankara.GFP (MVA) vector alone. Group2 mice received the same dose i.v. of recombinant MVA.Hav expressing theHavilah construct. At the end of the study 10 days later the mice werekilled using a humane procedure. Spleen weights were recorded and spleencells were obtained.

Stimulation of Splenocytes and ELISPOT Assay.

Spleen cells were harvested into 5 ml of RPMI (Sigma, UK) supplementedwith 2 mM glutamine, 1× penicillin-streptomycin (from a 100× stock, LifeTechnologies, UK) and 10% FCS (Life Technologies, UK). Cells werestrained using 70 μM cell strainer (BD biosciences) and pelleted bycentrifugation 200 g for 5 minutes at 4° C. Erythrocytes were lysedusing 1 ml of Red Cell Lysis Buffer (Sigma, UK) for 1 m at roomtemperature and neutralised with 14 ml of RPMI. Cells were washed withRPMI twice by centrifugation as described and resuspended in 2 ml ofRPMI with supplements. 500 of each pool of previously preparedrecombinant AhpC or MPA antigen diluted in RPMI and supplements wereadded to the wells of 96 well PVDF membrane filter plates (cat#S2EM04M99, Millipore, UK), which were previously coated with captureantibody. 50 μl of splenocytes adjusted to a concentration of 2.5×10⁷cells/ml were added to the wells containing antigen and incubated. Finalconcentration of antigens in each well was 2.5 μg/ml of recombinantprotein. The following materials were obtained from BD BiosciencesPharmingen, UK. BD™ ELISPOT Horseradish peroxidase, BD™ AEC substrateset, and BD™ mouse gamma interferon cytokine ELISPOT pair consisting ofa capture and detection antibody. The ELISPOT procedure followed wasthat described in BD Pharmingen Technical data sheet TDS Mar. 24, 2003.Spots were enumerated manually. Statistical analysis was performed usingthe Mann-Whitney (two tailed) test.

Results.

No adverse effects were seen in either control Group 1 mice or theexperimental Group 2 mice during the course of the study or at autopsy.No significant difference was found in spleen weights between the twoGroups. Mean ELISPOT responses (FIG. 3A) by spleen cells to purifiedrec.AhpC antigen were 83.3 in sham vaccinated Group 1 and 903.8 in thetest vaccinated Group 2 (p<0.015). Mean ELISPOT responses by spleencells to purified rec.MPA antigen were 91.0 in sham vaccinated Group 1and 900.7 in the test vaccinated Group 2 (p<0.004) (FIG. 3A). Highlysignificant ELISPOT responses were also seen with peptide groups B and Fcompared with vector-only controls (FIG. 3A). In a further experiment(FIG. 3B) ELISPOT responses were determined for the individual peptideswithin pool F. The entire response is seen to be due to prominentrecognition of peptide 9.1. This has the sequence GFAEINPIA (FIG. 4) andcomprises a strong T cell epitope corresponding to the 5^(th)extracellular loop in mpa (FIG. 5A) and within Havilah Seq ID. No 24residues 761-769 and FIG. 1.

Conclusion.

Prime boost vaccination of female C57/BL6 mice using plasmid and MVAvectors expressing Havilah are highly immunogenic resulting insignificant populations of antigen specific spleen cells against bothAhpC at the amino terminus, and MPA at the carboxy terminus and withsynthetic peptide epitopes of the Havilah polyprotein. The high level ofantigen specific immunity to Havilah polyprotein and peptide epitopesfollowing vaccination as described is not associated with any adverseeffect.

3. Safety and Efficacy of Therapeutic and Protective Vaccination Againsta Slow Growing Laboratory Strain of MAP Using pSG2.HAV then MVA.HAV.

A first study was carried out to test the Havilah vaccine as a treatmentfor pre-existing Mycobacterium avium subspecies paratuberculosis (MAP)infection as occurs in Johne's disease in animals and Crohn's diseaseand Irritable Bowel Syndrome in humans. Four groups of 8 naïve 4-6 weekold C57/BL6 female mice were isolator maintained. Their physicalcondition was monitored daily and body weights recorded twice weekly andat the end of the study. After an initial settle-in period of 7 days all32 mice were infected with 4×10⁷ of a slow growing attenuated laboratorystrain of bovine MAP i.p. in 200 μl sterile saline. Four weeks latercontrol Group 1 mice were given 25 μg of pSG2 expression plasmid in 50μl sterile endotoxin free TE buffer i.m. into each thigh. Experimentalgroup 2 mice were prime vaccinated with 25 μg of rec.pSG2.Hav in 50 μlsterile endotoxin free TE buffer i.m. into each thigh. Control Group 3mice received 50 μl of sterile endotoxin free TE buffer i.m. into eachthigh. This priming procedure was repeated after 1 week. Nine days latercontrol Group 1 mice were given 5×10⁶ pfu Modified Vaccinia Ankara.GFP(MVA) vector alone i.v. into the tail vein in 100 μl sterile endotoxinfree TE buffer. Experimental Group 2 mice were boost vaccinated with thesame dose i.v. of recombinant MVA.Hav expressing the Havilah construct.Control Group 3 mice were given 100 sterile endotoxin free TE bufferi.v. into the tail vein. Pairs of naïve mice from Group 4 were autopsiedat intervals throughout the study to monitor the progress of the MAPinfection. The study was terminated 26 weeks after the original MAPinfection. The infective load of MAP organisms was measured in thespleens and livers of all the mice using a sensitive and specificquantitative real-time IS900 PCR.

A second study was carried out to test the ability of the Havilahvaccine to confer some protection against subsequent MAP infection.Three groups of 8 naïve 4-6 week old C57/BL6 female mice were isolatormaintained. Their physical condition was monitored daily and bodyweights recorded twice weekly and at the end of the study. After aninitial settle-in period of 7 days control Group 1 mice were given 25 μgof pSG2 expression plasmid in 50 μl sterile endotoxin free TE bufferi.m. into each thigh. Experimental group 2 mice were prime vaccinatedwith 25 μg of rec.pSG2.Hav in 50 μl sterile endotoxin free TE bufferi.m. into each thigh. Control Group 3 mice received 50 μl of sterileendotoxin free TE buffer i.m. into each thigh. This priming procedurewas repeated after 1 week. Ten days later control Group 1 mice weregiven 5×10⁶ pfu Modified Vaccinia.Ankara.GFP (MVA) vector alone i.v.into the tail vein in 100 μl sterile endotoxin free TE buffer.Experimental Group 2 mice were boost vaccinated with the same dose i.v.of recombinant MVA.Hav expressing the Havilah construct. Control Group 3mice were given 100 μl sterile endotoxin free TE buffer i.v. into thetail vein Eight days later all mice were infected with 4×10⁷ MAP bovinestrain K10 i.p. in 200 μl sterile saline. The study was terminated 24½weeks later and the infective load of MAP organisms was measured in thespleens and livers of all the mice using quantitative real-time IS900PCR as before.

Results.

No adverse effects of the vaccine were seen in any of the Havilahvaccinated or control mice throughout the study or at autopsy. In thetherapeutic vaccination study the numbers of MAP organisms in thespleens of the mice in control Groups 1 and 3 were generally in therange 10 to 10,000 per gram of tissue. In the vaccine treated Group 2 noMAP organisms could be detected at all in the spleens of 6 of the 8 mice(FIG. 5B). In the other 2 animals in this group the number of MAP wasaround the lower limit of detection of the qRT-PCR in the range 1-10 pergram of tissue p=0.003 for Group 2 versus controls. The infective loadof MAP in the liver of Havilah vaccine treated mice in Group 2 was alsosignificantly reduced p=0.019. In the study of protective vaccinationthe efficacy of the vaccine was not as dramatic as therapeuticvaccination but the infective load of MAP in the liver was significantlylower than control mice p=0.0074.

Conclusions.

Therapeutic vaccination against MAP using plasmid and MVA vectorsexpressing the Havilah construct results in a highly significantattenuation of the MAP infection. Vaccination using these vectorsexpressing the Havilah construct has a smaller but significantprotective effect against subsequent MAP infection. The pre-existence ofMAP infection appears to confer advantage by priming the response to thevaccine used in its therapeutic role. Vaccination in the presence of MAPinfection using vectors containing Havilah is safe and is not associatedwith any adverse effect.

4. Safety and Immunogenicity of Vaccination Using Ad5.HAV then MVA.HAV

A further study was carried out in 3 groups of six naïve 4-6 week oldfemale C57/BL6 mice following the format previously described. Group 1received 10⁸ pfu of Ad5 vector in 50 μl buffer intradermally (i.d) intothe pinna of the ear; group 2 received the same dose of Ad5.HAV i.d andgroup 3 received 50 μl buffered saline only. Two weeks later the groups1-3 were vaccinated with 10⁸ pfu MVA vector only, 10⁸ pfu MVA.HAV orbuffer i.d respectively. The animals were killed at 4 weeks notingclinical and autopsy conditions and body weights. Spleen cells and serumwere obtained to quantify immunological responses to vaccine antigens.

Results.

No adverse effects due to the vaccination were seen in any of theanimals. Significant recognition of rec.AhpC, rec.gsd and rec.mpa inELISPOT assays as well as of pooled peptide antigens especially peptide9.1 GFAEINPIA and pool J compared with controls, again occurred. Therewas also significant recognition of the recombinant antigens AhpC andmpa by antibody in the vaccinated group compared with control groups.

To test the reproducibility of these findings the study was repeatedusing 3 similar groups of eight mice and a dose of 2×10⁷ pfu of eitherviral vector alone or 2×10⁷ pfu Ad5.HAV followed by 2×10⁷ pfu MVA.HAVi.d in 50 μl into the pinna of the ear. The animals were killed after 4weeks.

There was no evidence of adverse effects in any of these animals. Immuneresponses shown in FIGS. 6A and B were the same as seen previously.There was again strong ELISPOT recognition of peptide 9.1 andsignificant responses to recombinant antigens and peptide pool Jcompared with vector only controls. By contrast there was no recognitionof peptide 9.1 or any of the peptide pools by antibody and substantialantibody recognition of recombinant antigens AhpC and mpa.

Conclusions.

Vaccination of uninfected female C57/BL6 mice using Ad5.HAV followed byMVA.HAV expressing Havilah is highly immunogenic resulting insignificant populations of antigen specific spleen cells recognizingboth recombinant antigens and synthetic peptide epitope pools. Ofparticular note is the repeated demonstration of the strong specific Tcell epitope GFAEINPIA comprising the 5^(th) extracellular loop of mparecognized in vaccinated mice. Antigen specific immunity to Havilahpolyprotein following vaccination is reproducibly not associated withany adverse effect.

5. Safety and Efficacy of Therapeutic and Protective Vaccination Againsta Virulent Recent Disease Isolate of MAP Using Ad5.HAV then MVA.HAV

Two further studies were carried out each using 2 or 3 groups of eight4-6 week old female C57/BL6 mice to determine the safety and efficacy ofvaccination against a fast growing virulent recent disease isolate ofbovine MAP using 2×10⁷ pfu Ad5.HAV then 2×10⁷ pfu MVA.HAV compared withcontrol groups receiving vector-only or buffered saline alone, followingthe format previously described. In a high dose study the animalsreceived 10⁷ MAP i.p and in a low dose study 10⁵ MAP i.p. The study wasterminated after 8 weeks of MAP infection and the infective load of MAPorganisms was measured in the spleens and livers of all the mice using asensitive and specific quantitative real-time IS900 PCR.

Results.

None of the animals demonstrated any adverse effects of vaccination. Thenumbers of MAP organisms in the spleen tissues of the mice vaccinatedtherapeutically or prophylactically was significantly reduced comparedto control groups both in the low dose and high dose studies (FIG. 7).With the exception of the high dose study the numbers of MAP organismsin the liver tissues of the mice vaccinated therapeutically orprophylactically was also significantly reduced compared to controlgroups (FIG. 8). FIG. 9 shows that the strong T cell epitope GFAEINPIAin peptide 9.1 is again prominently recognised despite the presence ofvirulent MAP infection. Peptide pool J is significantly recognised inboth prophylactic and therapeutic vaccination. Peptide pool L is againsignificantly recognised when vaccination is given to animals which arealready MAP infected consistent with the prior infection ‘priming’ theresponse to the vaccine used therapeutically.

Conclusions.

Vaccination using Havilah expressed in plasmid, Adenovirus and MVA poxvirus vectors given by different immunisation routes in different dosesdemonstrates a reproducible and highly significant antigen-specific Tcell immunogenicity in mice without adverse effect. Vaccination inducedsignificant T cell recognition both of recombinant Havilah proteins andof synthetic peptide epitopes within the Havilah sequence, especiallythe strong T cell epitope GRAEINPIA corresponding to the 5^(th)extracellular loop of the mpa moiety. Significant antibody responses torecombinant AhpC and mpa also occurred. Using different vectorcombinations in different closes and different routes of administrationagainst different strains of MAP, vaccination using Havilah constructsrepeatedly results in significant therapeutic attenuation ofpre-existing MAP infection and protection against subsequent MAPinfection. Furthermore, responses to therapeutic vaccination may beenhanced by the ‘priming’ effect of pre-existing MAP infection. Havilahmay be used as a vaccine to confer protection against MAP infections andto treat MAP infections of animals and humans such as Johne's diseaseand Crohn's disease and Irritable Bowel syndrome as well as topotentiate the clinical response to treatment with anti-MAP drugs.

1. A polypeptide comprising an ahpC polypeptide sequence, a gsdpolypeptide sequence, a p12 polypeptide sequence and an mpa polypeptidesequence, wherein said ahpC polypeptide comprises the sequence of SEQ IDNO: 2, a variant thereof having more than 70% amino acid sequenceidentity to SEQ ID NO: 2 across the full length of SEQ ID NO: 2, or afragment of at least 8 amino acids of SEQ ID NO: 2 which comprises anepitope; said gsd polypeptide comprises the sequence of SEQ ID NO: 6, avariant thereof having more than 70% amino acid sequence identity to SEQID NO: 6 across the full length of SEQ ID NO: 6, or a fragment of atleast 8 amino acids of SEQ ID NO: 6 which comprises an epitope; said p12polypeptide comprises the sequence of SEQ ID NO: 10, a variant thereofhaving more than 70% amino acid sequence identity to SEQ ID NO: 10across the full length of SEQ ID NO: 10, or a fragment of at least 8amino acids of SEQ ID NO: 10 which comprises an epitope; and said mpapolypeptide comprises the sequence of SEQ ID NO: 14, a variant thereofhaving more than 70% amino acid sequence identity to SEQ ID NO: 14across the full length of SEQ ID NO: 14, or a fragment of at least 8amino acids of SEQ ID NO: 14 which comprises an epitope.
 2. Apolypeptide according to claim 1 wherein said ahpC polypeptide has theamino acid sequence given in SEQ ID NO:
 4. 3. A polypeptide according toclaim 1 wherein said gsd polypeptide has the amino acid sequence givenin SEQ ID NO:
 8. 4. A polypeptide according to claim 1 wherein said p12polypeptide has the amino acid sequence given in SEQ ID NO:
 12. 5. Apolypeptide according to claim 1 wherein said mpa polypeptide has theamino acid sequence given in SEQ ID NO:
 16. 6. A polypeptide accordingto claim 1 wherein said mpa polypeptide comprises the amino acidsequence GFAEINPIA.
 7. A polypeptide according to claim 1 whichcomprises the amino acid sequences of SEQ ID Nos: 4, 8, 12 and
 16. 8. Apolypeptide according to claim 1 which comprises the amino acid sequencegiven in SEQ ID NO:
 24. 9. A polynucleotide which encodes a polypeptideaccording to claim
 1. 10. A polynucleotide according to claim 9 whichcomprises (a) the ahpC polynucleotide of SEQ ID NO: 1 or a variantthereof having at least 70% homology to SEQ ID NO: 1 across the fulllength of SEQ ID NO: 1 or a fragment of at least 24 nucleotides of SEQID NO: 1 which encodes an epitope; (b) the gsd polynucleotide of SEQ IDNO: 5 or a variant thereof having at least 70% homology to SEQ ID NO: 5across the full length of SEQ ID NO: 5 or a fragment of at least 24nucleotides of SEQ ID NO: 5 which encodes an epitope; (c) the p12polynucleotide of SEQ ID NO: 9 or a variant thereof having at least 70%homology to SEQ ID NO: 1 across the full length of SEQ ID NO: 9 or afragment of at least 24 nucleotides of SEQ ID NO: 9 which encodes anepitope; and (d) the mpa polynucleotide of SEQ ID NO: 13 or a variantthereof having at least 70% homology to SEQ ID NO: 1 across the fulllength of SEQ ID NO: 13 or a fragment of at least 24 nucleotides of SEQID NO: 13 which encodes an epitope.
 11. A polynucleotide according toclaim 10 wherein said ahpC polynucleotide has the sequence given in SEQID NO:
 3. 12. A polynucleotide according to claim 10 wherein said gsdpolynucleotide has the sequence given in SEQ ID NO:
 7. 13. Apolynucleotide according to claim 10 wherein said p12 polynucleotide hasthe sequence given in SEQ ID NO:
 11. 14. A polynucleotide according toclaim 10 wherein said mpa polynucleotide has the sequence given in SEQID NO:
 15. 15. A polynucleotide according to claim 10 which comprisesthe nucleic acid sequences of SEQ ID Nos: 3, 7, 11 and
 15. 16. Apolynucleotide according to claim 10 which comprises the nucleic acidsequence given in SEQ ID NO:
 24. 17. A vector comprising apolynucleotide according to claim
 9. 18. A vector capable of expressingan ahpC polypeptide, a gsd polypeptide, a p12 polypeptide and an mpapolypeptide as defined in claim
 1. 19. A vector according to claim 17which is a poxvirus vector, an adenovirus vector or a plasmid.
 20. Ahost cell comprising a vector according to claim
 17. 21. A host cellwhich is capable of expressing a polypeptide according to claim
 1. 22. Amethod of treating or preventing MAP infection or a condition or symptomassociated with MAP infection comprising administering to a subject inneed thereof an effective amount of a polynucleotide according to claim9.
 23. A kit for use in treating or preventing MAP infection or acondition or symptom associated with MAP infection, said kit comprising(i) at least one polynucleotide according to claim 9 and (ii) at leastone other therapeutic agent, for simultaneous, sequential or separateuse.
 24. A method according to claim 22 wherein said method is fortreating or preventing chronic inflammation of the intestine,inflammatory bowel disease, Irritable Bowel Syndrome, chronic enteritis,Johne's disease or Crohn's disease.
 25. A method according to claim 22wherein the individual being treated is also administered a furthertherapeutic agent which has activity against MAP or a furthertherapeutic agent used in the treatment of a condition which isassociated with MAP infection.
 26. A method of treating or preventingMAP infection or a condition or symptom associated with MAP infectioncomprising administering to a subject in need thereof an effectiveamount of a polypeptide according to claim 1.